The potential of phenothiazinium dyes as cytotoxicity markers in cisplatin-treated cells

Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the strategies employed to determine cytotoxicity, MTT, neutral red, and resazurin are commonly used. Methylene blue (MB), a phenothiazinium salt, has several uses, such as dye, redox indicator, and even as treatment for human disease and health conditions, such as malaria and methemoglobinemia. However, MB has only been sparsely used as a cellular toxicity indicator. As a viability indicator, MB is mostly applied to fixed cultures at high concentrations, especially when compared to MTT or neutral red. Here we show that MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) can be used as cytotoxicity indicators in live (non-fixed) cells treated for 72 h with DMSO and cisplatin. We compared dye uptake between phenothiazinium dyes and neutral red by analyzing supernatant and cell content via visible spectra scanning and microscopy. All dyes showed a similar ability to assess cell toxicity compared to either MTT or neutral red. Our method represents a cost-effective alternative to in vitro cytotoxicity assays using cisplatin or DMSO, indicating the potential of phenothiazinium dyes for the screening of candidate drugs and other applications.

The approval of new molecules for use in humans and animals depends on the evaluation of many properties, one of the most critical of which is toxicity. Assessment of toxicity for candidate compounds is initially performed via incubation with mammalian cells 1 . Then, several parameters pertaining to cell integrity, cell viability, and/ or metabolism are evaluated [2][3][4] . Vital dyes, as the name implies, accumulate in live cells and allow the measuring of cell viability. For instance, trypan blue, methylene blue (MB), erythrosine B, nigrosine, eosin, safranin, propidium iodide, and 7-aminoactinomycin D have been applied for the determination of cell viability for diverse purposes 5 . Additionally, there are indicators that measure the activity of metabolic pathways related to cell viability; these are exemplified by the tetrazolium salts MTT, XTT, WST-1, and MDS, which have also been widely used for cell viability assays. For example, MTT is reduced by mitochondrial dehydrogenases in viable cells to MTT formazans, which are spectrophotometrically quantified at 570 nm 6,7 . Similarly, resazurin (Alamar blue) is also used for cell viability assays, in which the molecule (a blue dye) is reduced to the highly fluorescent resorufin by intracellular diaphorase enzymes 8,9 . Molecules incorporated by live cells are also used as indicators of toxicity in drug screening strategies. For example, neutral red (NR) is a dye that is protonated (ionized) at low pH and thus accumulates in lysosomes due to the low pH of these organelles. Therefore, drugs that interfere with cell membranes and/or lysosomes decrease NR uptake, allowing the dye to serve as a cell viability indicator 10,11 . In this sense, MB is also applied as an indicator of cytotoxicity in mammalian cell culture because it accumulates in non-damaged cells. However, MB is mostly used as a cell viability indicator after formalin fixation and, for this application, is also used at higher concentrations (1% w/v) compared to NR (50 µg/ml; 0.005% w/v) 12,13 .
In the present study, we evaluated a method based on the accumulation of MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) in live cells after treatment with DMSO and cisplatin. Cisplatin or cis-diamminedichloridoplatinum(II) is one of the most potent and widely used drugs for the treatment of solid tumors 14 . The most accepted mechanism of cisplatin is based on its interaction with purine bases, which results in DNA lesions leading to cell apoptosis 15 . However, side effects and drug resistance are constant challenges of cisplatin treatment 16,17 . This underscores the importance of novel strategies such as drug modifications or combinations to minimize side effects 18,19 . In this sense, the development of new cisplatin therapies depends on in vitro cytotoxicity assays, especially those with different mechanisms of detection that are capable of complementing classic methods (i.e. MTT, NR, and resazurin). Here we evaluated the potential of phenothiazinium dyes as indicators of cytotoxicity in cisplatin-sensitive and -resistant cell lineages. After 72 h of cisplatin treatment, phenothiazinium dyes showed a similar pattern of cell viability compared to MTT and NR. The use of phenothiazinium dyes for the evaluation of cisplatin cytotoxicity indicates an alternative, efficient, and cost-effective method for in vitro cell viability assays.

Detection of the phenothiazinium dyes accumulation in cells.
The accumulation of phenothiazine dyes in cells was detected by light and confocal microscopies. Firstly, Vero cells were incubated with MB, NMB, TBO, and DMMB (100, 50, 25 and 12.5 µM) for 3 h, 37 °C, 5% CO 2 and washed with PBS. The cells were observed in a light microscope and representative pictures of each incubation regimen acquired. For confocal detection of phenothiazinium dyes, Vero cell cultures were incubated with 10 μM of MB, NMB, TBO or DMMB for 30 min, 37 °C, with 5% CO 2 . For all procedures, non-treated controls were incubated with RPMI under the same conditions. The cultures were washed with PBS and treated with trypsin for 10 min, 37 °C and 5% CO 2 . The cells were transferred to slides and analyzed by confocal microscopy with excitation/emission wavelengths of 543/600 nm, respectively. The slides were observed in a TCS-SP8 AOBS (Leica Microsystems), using a 63× objective and processed by ImageJ software (version 1.53j, National Institute of Health, USA).
Calculation of the logarithm of the octanol-water partition coefficient (log P) and the negative logarithmic of the acid dissociation constant (pK a ). To predict lipophilicity and localization of the phenothiazinium dyes in cells, we used log P and pK a values as described in the literature 21,22 . Thus, log P was estimated using the method proposed by Ref. 23 , as defined by Ref. 24 as being the "sum of the log P of the parent solute plus a π term" (Eqs. 1, 2). Reference 25 defined that the rate of penetration of a given species to an active site is a parabolic function, that could be simplified by using π, a substituent constant derived from the partition coefficients, defined numerically as: Therefore, by adding π values to a log P, we have: Besides the calculations applying Eq. (2) and experimentally obtained π values, presented too by Ref. 24 , we also resorted to in silico calculations of log P, pK a and log D of MB, NMB, TBO and DMMB using ChemAxon's MarvinSketch (version 23.5) and its protonation and lipophilicity calculators. The molecular structures adopted were those for which the optimization is available at PubChem (https:// pubch em. ncbi. nlm. nih. gov/). ChemDraw (version 21.0.0) was also used to draw the molecules shown in the text. The reference for pH values at physiologic media, lysosomes and mitochondria was obtained from Ref. 26 .
Visible absorption spectra of phenothiazinium dyes and their cell uptake. Initially, to evaluate the absorption of dyes by cells, MB, NMB, TBO, and DMMB were diluted in phenol red-free RPMI (100 µM) www.nature.com/scientificreports/ and incubated with Vero cell monolayers (in 24-well plates) for 4 h at 37 °C and 5% CO 2 . An aliquot (200 µl in duplicate) of the supernatants were collected at intervals of 1 h (0, 1, 2, 3, and 4 h) and divided in two equal parts: the first part was homogenized with an alcohol-acid solution (2% acetic acid in absolute ethanol) and the second part was used as control. After incubation with phenothiazinium dyes, cell monolayers were washed with 100 µl of fixing solution (1% CaCl 2 and 0.5% formaldehyde), followed by dye extraction with 250 µl of extraction solution (1% acetic acid in 50% ethanol). All samples (supernatants and cell extracts) were analyzed via visible light spectrophotometry in an ELISA reader (Epoch Biotek) and the area under the spectral curve (cell extracts) was calculated (using the absorbance values from 380 to 800 nm). The values from the incubated samples (1, 2, 3, and 4 h) were used to calculate the percentage of dye compound uptake relative to the initial absorbance (0 h). Three independent experiments were performed.
Sensitivity assay for the detection of phenothiazinium dyes in Vero cells. Vero cells were distributed in 96-well plates and cultivated until confluence was achieved. Then, cells were incubated with 10 serial dilutions of MB, NMB, TBO, and DMMB (starting at 250 µM) for 3 h at 37 °C and 5% CO 2 . After incubation, cells were fixed with 100 µl of fixing solution and incorporated dye was extracted with 100 µl extraction solution. The detection of phenothiazinium dyes were performed in an ELISA reader at 660 nm, 630 nm, 630 nm and 650 nm for MB, NMB, TBO, and DMMB, respectively. The difference between the absorbance values of the dilutions and the control (composed of free dye RPMI) was applied as a sensitivity indicator of dyes in cells. Two independent experiments were performed.

Results
Accumulation of phenothiazinium dyes in Vero cells. Cells incubated with phenothiazines at 100 µM displayed no sign of toxicity and were morphologically similar to the control group (Fig. 1A). We observed that uptake of phenothiazinium dyes was diverse for each dye tested. On the one hand, MB and TBO had a heterogeneous accumulation pattern: stained cells presented spots with high dye content and the number of unstained cells was higher ( www.nature.com/scientificreports/ Detection of phenothiazinium dyes by fluorescence showed their accumulation in the cell cytoplasm. The dyes were detected by fluorescence in vesicle-like structures, which were not observed in untreated control ( Fig. 2A). Among the dyes, MB showed less accumulation relative to NMB, TBO, and DMMB ( Fig. 2B-F).

Lipophilicity prediction and phenothiazinium dyes localization within cells.
The values for log P were calculated either by applying Eq. (2) 24 or by in silico ChemAxon calculators. Both methods resulted in the same pattern of dye lipophilicity: MB and TBO displayed a more hydrophilic behavior, with negative log P values, whereas NMB and DMMB had positive values, indicating a more lipophilic profile (Tables 1, 2). The use of Eq. (2) resulted in log P values for MB and TBO of − 0.62 and − 1.47, respectively (Table 1). Although in silico calculations presented slightly different numbers, the same overall pattern remained, with log P values of − 0.51 (MB) and − 1.09 (TBO) ( Table 2). For DMMB, log P values obtained using Eq. (2) and the ChemAxon calculator were 0.5 and 0.23, respectively (Tables 1, 2). According to in silico estimations, among the phenothiazinium dyes, NMB is the only one that can have a free base form within the physiological pH range, in addition to the protonated molecule (Table 1). Adopting Eq. (2), the free base form of NMB has a log P of 4.84, while the software calculated a log P of 3.55. This happened because the last also considers the contribution of protonated forms to estimate log P. If one takes a closer look at π values of protonated amines 24 , it is easier to understand why they lower log P: the radical + N(CH 3 ) 3 contributes with a π value of − 5.96, for example. When Eq. (2) was applied, the obtained log P for the protonated form of NMB decreased to 0.4 ( Table 1). The predicted pK a values of MB (~ 3.14), TBO (~ 3.17), and DMMB (~ 3.86) indicate a higher prevalence of protonated forms in physiological, lysosomal, or mitochondrial pHs ( Supplementary Fig. 2). The pK a of NMB (7.03) indicates a coexistence between the protonated and free base forms in acid and alkaline solutions, respectively ( Supplementary  Fig. 2). The lipophilicity tendencies were maintained for all dyes in physiologic (pH 7.4), lysosomal (pH 4.7) and mitochondrial (pH 8.0) milieus. In all analyzed conditions, the log D (the calculated lipophilicity when standard conditions are not met, in this case, in a specific pH) values were negative for MB and TBO and positive for NMB and DMMB ( Table 2).
Detection of phenothiazinium dyes in the culture supernatant. All phenothiazinium dyes diluted at 100 µM were promptly detected in the visible spectrum (Fig. 3). MB in RPMI presented two maximum absorp- www.nature.com/scientificreports/ tion peaks (610 and 660 nm), which became a single peak at 660 nm after the addition of an alcohol-acid solution (Fig. 3A,B). Conversely, both NMB and TBO (in RPMI) produced a single peak at 580 nm, which shifted to 630 nm in an alcohol-acid solution (Fig. 3C-E). DMMB also presented a peak shift, but in this case from 570 to 650 nm (Fig. 3G,H). All phenothiazinium dyes were readily absorbed/internalized by the cells, leading to a decreased spectral absorption in the supernatant over time (Fig. 3A-H). Also, the dyes were incorporated by cells within the first hour of incubation, with a lower rate of absorption observed during the second, third, and fourth hours of culture (Fig. 3A-H). The two spectral peaks, characteristic of MB in RPMI, decreased from 1.0-1.1 to 0.5-0.6 after 1 h of incubation. Similarly, when the same supernatant was diluted in an acid-alcohol solution, the peak at 660 nm reduced from 1.7 to 1.2 (Fig. 3A,B). The peaks of detection of NMB, TBO, and DMMB in RPMI decreased from 1.30, 0.95, and 0.50 to 0.55, 0.50, and 0.18, respectively, after 1 h of incubation (Fig. 3C,E,G). A similar pattern was observed in supernatants diluted in an alcohol-acid solution. The peaks of detection of NMB, TBO, and DMMB reduced from 2.45, 1.70, and 1.60 to 1.20, 0.90, and 0.50, respectively (Fig. 3B,D,F,H). In general, after the second, third, and fourth hours of incubation, the absorbance rates for the compounds were lower for MB and TBO compared to NMB and DMMB. The maximum absorption peak for Table 1. Names, chemical structures and estimated log P of phenothiazinium dyes calculated using Eq. (2).   www.nature.com/scientificreports/ NR in RPMI varied by fewer than 0.2 units (Fig. 3I) whereas the sample diluted in alcohol-acid decreased from 1.6 to 0.9 (Fig. 3J).

Detection of intracellular phenothiazinium dyes in cell monolayers. Phenothiazinium dyes that
accumulated in cell monolayers were extracted with NR extraction solution after fixation. During incubation, intracellular dye concentrations increased, mainly after the first and second hours of incubation (Fig. 4). Similar to the observed for the supernatant analyses, the phenothiazinium dyes presented higher levels of internalization compared to NR. Uptake of NMB and DMMB was higher compared to MB or TBO. To quantify this phenomenon, we calculated the area under the absorbance curve for each dye used. The areas under the curve for NMB were 14.3, 17.3, 19.9 and 20.5 after 1, 2, 3 and 4 h of incubation, whereas those for MB and TBO were 8.25/10.63, 9.84/11.87, 10.38/13.58 and 12.10/13.97, respectively (Fig. 5). For DMMB, the area under the curve was 13.3 (1 h), 17.7 (2 h), 19.6 (3 h) and 21.9 (4 h). Areas under the curve for NR were between 5.5 and  www.nature.com/scientificreports/ 10.96 (after 1 and 4 h of incubation, respectively), with these values being lower than those observed for all phenothiazines (Fig. 5).

Sensitivity of phenothiazinium dyes detection in cells.
The minimum limit for detection of phenothiazinium dyes varied according to the dye: MB, NMB, TBO, and DMMB were detected in concentrations above 31.2, 7.8, 15.6, and 7.8 µM, respectively (Fig. 6). Except for TBO, correlation between absorbance and dye concentration was good for all phenothiazines (Fig. 6). For TBO, the absorbance/concentration correlation was observed only until 62 µM (Fig. 6). A low difference in detection among phenothiazines was observed at 125 µM, a concentration for which absorbance varied from 0.29 (TBO) to 0.53 (DMMB). At 100 µM (calculated using the linear regression equation), the difference in absorbance among dyes was lower compared to 125 µM, corresponding to 0.34, 0.39, 0.35, and 0.50 for MB, NMB, TBO, and DMMB, respectively (Fig. 6). Therefore, a concentration of 100 µM was selected for an adequate comparison among the dyes in the experiments that followed (i.e., linearity and cytotoxicity assays).

Linearity of phenothiazinium dyes in Vero cells. The accumulation of phenothiazinium dyes in Vero
cells was proportional to cell numbers within each well. All dyes were detected at cell densities above 10 3 cells/ well, similar to what was observed for MTT and NR (Fig. 7). All compounds had consistent correlation between cell number and absorbance, reaching saturation at densities above 1 × 10 4 cells/well. Among the compounds, NMB and TBO showed dye saturation at 2.5 × 10 4 cells/well whereas MB, DMMB, MTT, and NR reached a peak of detection at 1.25 × 10 4 cells/well (Fig. 7).   www.nature.com/scientificreports/ Phenothiazinium dyes as indicators of cytotoxicity in cells. Phenothiazinium dyes were tested as cytotoxicity markers and their performance was measured against that of NR and MTT. Initially, Vero cells and fibroblasts were incubated with different concentrations of DMSO and cell viability was assessed. Vero cells and fibroblasts were inhibited (> 70%) by DMSO in concentrations above 5%, with decreased inhibition for more diluted samples (< 5%). Nonetheless, DMSO maintained a basal cell inhibition at concentrations of 2.5% and lower, especially as measured by TBO (Fig. 8). Furthermore, phenothiazinium dyes allowed the evaluation of cisplatin cytotoxicity in both resistant and susceptible cell lines. After incubation with cisplatin, staining intensity of the toxicity markers showed a dose-response profile. Similar intensity patterns were observed between phenothiazinium (especially NMB, TBO, and DMMB) and NR. Monolayers of MDA-MB-231 cells (Fig. 9A) showed less intense staining than MCF 10A (Fig. 9B) and LLC-MK2 (Fig. 9C) cells.
Quantification of the apparent staining changes resulted in the same pattern (Fig. 10). At concentrations above 12.5 µM, MDA-MB-231 cells were inhibited by more than 50% (Fig. 10A). On the other hand, MCF 10A and LLC-MK2 cells were resistant to cisplatin at concentrations below 25 µM and 50 µM, respectively (Fig. 10B,C). As observed on 96-well plates (Fig. 9B), the MCF 10A cell line had higher tolerance to cisplatin at 200 µM compared to 100 µM (Fig. 10B). For 100 µM and above, the curve fit of MCF 10A cells followed a dose-response pattern, similar to the observed for LLC-MK2 cultures (Fig. 10B,C). All inhibition curves from cell lines incubated with dyes allowed the calculation of IC 50 values.
The IC 50 values followed similar patterns for the phenothiazinium dyes, NR, and MTT. Vero cells and fibroblasts had equal susceptibility to DMSO, with IC 50 values between 3.65 (for NR) and 2.76 µM (for MB) (Fig. 11A) (Fig. 11B). There was no statistically significance difference between the performance of phenothiazines and NR or MTT for the quantification of cytotoxicity in the presence of DMSO and cisplatin.

Discussion
The uptake of phenothiazinium dyes by viable cells revealed their potential as markers of cytotoxicity. Moreover, these dyes presented high stability, low toxicity, low cost, and simple and safe handling. Such factors, together with good assay reproducibility, are crucial factors for the development of methods to assess the in vitro cytotoxicity of compounds 32 . Each phenothiazinium dye showed a different absorption pattern by cells, especially when MB and TBO were compared to NMB and DMMB. However, all dyes had tropism for the cell cytoplasm, especially when diluted. Indeed, MB has affinity for lysosomes as previously reported for both cancerous and normal breast tissue cell lines 33 . In general, phenothiazinium dyes incubated with cells are absorbed within the first hour of incubation, similarly to what is observed for NR. This fast absorption rate is an important factor for cell labeling because NR is toxic to cells 34 . Phenothiazinium dyes are also toxic to Vero cells in longer regimens of incubation. After 72 h of incubation, MB had low cytotoxicity at 100 µM (IC 50 > 62.5 µM), whereas NMB and TBO were toxic at lower concentrations (~ 20-25 µM). For DMMB, the toxic effects in Vero cells were observed www.nature.com/scientificreports/ at concentrations below 10 µM 30 . Thus, we tested the phenothiazinium dyes as cytotoxicity markers in short incubation periods (< 4 h), similar to the ones applied to NR or MTT assays. The phenothiazinium dyes presented several advantages over NR, such as higher solubility and increased stability in culture media. NR usually precipitates in culture, requiring filtration and/or overnight incubation in a refrigerator before use 34,35 . Other strategies applied to decrease NR precipitation are filtration of the medium at the time of use or pre-filtering of the dye stock, both of which usually reduce the discrepancy between assays [35][36][37] . Therefore, the use of NR as a cytotoxicity marker requires additional preparation steps compared to phenothiazinium dyes. Moreover, the precipitation of NR is increased in the presence of salts and/or fetal bovine serum: stock solutions of NR revealed a higher number of crystals in PBS compared to distilled water 38 . Conversely, no precipitation or crystallization were observed for phenothiazinium dyes at 100 µM. Additionally, the stock solutions of phenothiazinium dyes are stable in water for long periods (> 6 months) at 5 mg/ml.
The main difference between currently used cytotoxicity assays employing NR and MB is in the cell model of stain. NR is applied to living cells, which accumulate the dye in lysosomes 11 . In contrast, MB is used on formalin-fixed cells 39,40 . The method presented here is novel in that it uses viable cells, similar to NR. Indeed, the phenothiazinium dyes accumulated in vesicle-like structures in Vero cells. The MB accumulation is also observed for MB and TBO in HeLa cells 41 . Moreover, MB is reported to accumulate in the lysosomes of mammary cells 33 . www.nature.com/scientificreports/ Furthermore, our assay employs smaller amounts of phenothiazinium dyes compared to currently used methods applied to fixed cells. After fixation, MB labeling usually uses 5 to 10 g of dye per liter of staining solution 12,40 . Conversely, our study uses 52 mg/l of dye, similar to the concentration employed for NR (50 mg/l) 29,42 . Therefore, our results indicate a novel application of phenothiazinium dyes as viability markers.
In addition, we observed differences in absorption among the dyes: uptake by cells was more intense for NMB and DMMB, both of which equally stained the cultures. Conversely, MB and TBO presented a slower rate of dye incorporation compared to NMB and DMMB, producing an irregular pattern of dye accumulation. These differences among phenothiazinium dyes are a direct consequence of their chemical structures. Some parameters related to the molecular structure, such as log P and pK a can indicate the pattern of uptake and accumulation in cell compartments. We used two strategies to predict the log P values (manually calculated through Eq. (2) and in silico using ChemAxon calculators) with similar results. In general, MB and TBO showed a more hydrophilic profile (log P < 1), whereas NMB and DMMB had a more lipophilic nature (log P > 1) 43 . Thus, the positive log P values corroborate the faster uptake rate of NMB and DMMB when compared to MB and TBO. Moreover, MB and TBO are protonated at pH 7.4, resulting in a slower cell uptake relative to NR and NMB. In this sense, the MB and TBO accumulation in lysosomes is probably dependent on the extent of cell endocytosis. Indeed, the use of membrane carriers (i.e. liposomes or nanoparticles) has improved MB uptake by cells 44,45 through endocytosis pathways. For example, MB combined with citrate-coated maghemite nanoparticles (MAGCIT-MB) was internalized in breast and ovarian cell lines by the clathrin endocytosis pathway 46 . Once into the cells, hydrophilic dyes with ionizable amines (TBO) probably accumulate in lysosomes by ion trapping, similarly to the observed for NR. In this case, the cationic form of NR (pK a of 6.9) is membrane impermeant with a log P of − 1. However, under physiologic conditions, half of the NR molecules are in a free base state (log P = 1.9), which passively enter www.nature.com/scientificreports/ the cells. In lysosomes (pH ~ 4.5), the cationic form of NR is predominant, resulting in accumulation within these organelles via ion-trapping 21 . Our results indicated that the free base form of NMB is predominant in alkaline media (pH > 7.0). In mitochondria (pH ~ 8.0), approximately 89.7% of NMB corresponds to the free base form, resulting in higher lipophilicity values (log P 3.84) compared to MB and TBO. Indeed, the log P values of cationic probes between 0 and 5 indicate an affinity to mitochondria, as reported by Refs. 22,47 . Besides the cell uptake rates, NMB and DMMB also have different patterns of cell accumulation compared to MB and TBO. Despite current knowledge and advances, further assays may contribute to the comprehension of phenothiazinium dye uptake and distribution into cells. For example, co-localization studies with well-established organelle markers (LysoTracker™ and MitoTracker™) may better indicate the localization of MB, NMB, TBO and DMMB in cells. These differences among phenothiazinium dyes are also observed in other models. For example, DMMB and NMB exhibited higher toxicity in LLC-MK2 and Vero cells, in addition to parasiticidal activity against Neospora caninum and Trypanosoma cruzi, compared to MB and TBO 30,48 . Thus, further development of phenothiazinium analogues by manipulation of dye structure will potentially result in cytotoxicity markers with higher labeling capacity.
The differences among phenothiazinium dyes were also observed in sensitivity and linearity assays. In general, dyes incubated with Vero cells showed a good correlation between absorbance and dye concentration or cell density. NMB and TBO showed linearity with a higher number of cells (2.5 × 10 4 cells/well) compared to MB, DMMB, NR and MTT (1.2 × 10 4 cells/well). Probably, NMB and TBO have a lower saturation capacity compared to the other markers. However, new linearity assays using different cell lineages, dye concentrations, counters (i.e., flow cytometer) and markers (i.e. resazurin, LDH assay) may elucidate the differences in saturation among dyes. NMB and DMMB were detected at lower concentrations (> 7.8 µM) compared to MB and TBO (> 15.6 µM and > 31.2 µM, respectively). Although NMB and DMMB are the most sensitive, MB produced the highest absorbance value at 250 µM. Indeed, MB had a lower rate of accumulation, reaching saturation only at higher concentrations. However, in concentrations above 100 µM, all dyes have displayed toxic effects in cells (cell detachment, data not shown), indicating that the use of dyes at concentrations higher than 100 µM should be assessed in further assays. As such, we selected the concentration of 100 µM for cytotoxic assays. At this concentration, all dyes had low and non-significant affinity to the plastic material of plates (data not shown).
Furthermore, the dyes displayed linearity between absorbance and cell density, similar to the observed for MTT or NR. The values of r 2 for all phenothiazinium dyes tested were above 0.8, which are in accordance with www.nature.com/scientificreports/ previous studies using MTT or NR [49][50][51] . A linear correlation between cell density and phenothiazinium dyes, MTT or NR absorbance values was achieved for cell densities ranging from 1000 to 25,000 cells/well. At 12,500 cells/well (NR and MTT) or 25,000 cells/well (NMB and TBO) the curves were out of the linear range, which was also reported in studies using different cell lineages [52][53][54] . In general, our results indicate a correlation between dye accumulation and cell density, similar to the observed for assays based on formalin fixed assays 39,40 . However, the mechanism of dye accumulation is different in live and fixed cells. In formalin-fixed cells, MB has an affinity to negatively charged structures of the cell (i.e., DNA) 55 . In our model, using non-fixed (live) cultures, the dyes (mainly NMB and DMMB) have an affinity to vesicles like-structures similar to the observed for NR. Despite the correlation between dye accumulation and cell density, new assays concerning MB incorporation and cell viability are necessary. For example, the comparison between phenothiazinium dyes and vital dyes (propidium iodine or trypan blue) may indicate a future application of MB and analogues as cell viability markers.
To evaluate to which extent phenothiazines could be compared to NR and MTT as cytotoxicity markers, we used DMSO and cisplatin, which have been extensively used in cytotoxicity assays. DMSO is widely applied with low toxicity for solubilization of non-polar compounds for in vitro assays 56,57 . DMSO usually presents similar toxicity for diverse cell lines, mainly when applied at concentrations above 2% (v/v) 58,59 . Likewise, in our study, DMSO inhibited Vero cells and fibroblasts between 1.75 and 3.65%. The assays employing DMSO indicate the applicability of phenothiazines as indicators of cytotoxicity, showing similar patterns compared to MTT or NR. The similarity among phenothiazinium dyes, MTT, and NR was confirmed via assays with cisplatin applied to both susceptible and resistant cell lines. Cisplatin is an antitumoral drug used for the treatment of bladder, head and neck, lung, ovarian, and testicular neoplasms 60 . Several studies have reported the use of cisplatin for in vitro assays in several cell lines 61,62 . Our results indicated three patterns of susceptibility to cisplatin, according to cell line: MCF 10A is a line applied as resistance control (for mammary cancer) to cisplatin, with IC 50 concentrations between 5 and 26 µM 63,64 . In this study, MCF 10A was applied as non-cancerous control for the MDA-MB-231 cells, which is a breast adenocarcinoma lineage. Cisplatin was shown to inhibit 50% of the growth of MDA-MB-231 at 6 µM 63 , which is a similar value to the ones we observed for MTT, NR or the phenothiazinium dyes. To reinforce the reproducibility of our results, we tested the LLC-MK2 cell lineage, which is widely used in cytotoxicity assays 65,66 , including phenothiazinium dyes 48 . LLC-MK2 is a non-cancer cell lineage of renal tissue origin, with high robustness for cultivation [67][68][69] . The resistance and robustness of LLC-MK2 were confirmed by our results, which indicated the highest resistance to cisplatin compared to MCF 10A and MDA-MB-231 lines. Therefore, our study indicates a common pattern of susceptibility to cisplatin in different cell lineages, which validates the use of phenothiazinium dyes as cytotoxicity markers.
We have tested the cytotoxicity of cisplatin or DMSO in 72-h incubation regimens, which are applied in several drug screening strategies such as viral, parasitic and tumor models [70][71][72] . Cisplatin and DMSO are very different agents with clearly distinct modes-of-action. Nonetheless, our method was capable of evaluating their intrinsic toxicity to same extent as the classical assays. In this sense, our results open the perspective of developing novel assays for toxicity monitoring with varied molecules, from antimicrobials to antitumoral agents. In conclusion, this work presents the potential of phenothiazinium dyes as markers for cytotoxicity assays in living cells via a simple, easy-to-use, and cost-effective method that can be applied to the cytotoxicity assessment of candidate drugs in other models.

Data availability
All data generated during this study are included in this published article and its Supplementary Information files.